Masaki Mizunuma

Role of calcineurin and Mpk1 in regulating the onset of mitosis in budding yeast.

Signalling via calcium is probably involved in regulating eukaryotic cell proliferation, but details of its mechanism of action are unknown,. In Schizosaccharomyces pombe, the onset of mitosis is determined by activation of a complex of the p34cdc2 protein kinase and a cyclin protein that is specific to the G2 phase of the cell cycle. This activation requires dephosphorylation of p34cdc2 (ref. 3). Wee1, a tyrosine kinase that inhibits p34cdc2 by phosphorylating it, is needed to determine the length of G2 phase. Here we show that calcium-activated pathways in Saccharomyces cerevisiae control the onset of mitosis by regulating Swe1, a Wee1 homologue. Zds1 (also known as Oss1 and Hst1) (refs 4–7) is important in repressing the transcription of SWE1 in G2 phase. In the presence of high calcium levels, cells lacking Zds1 are delayed in entering mitosis. Calcineurin and Mpk1 (refs 12, 13) regulate Swe1 activation at the transcriptional and post-translational levels, respectively, and both are required for the calcium-induced delay in G2 phase. These cellular pathways also induce a G2-phase delay in response to hypotonic shock.

Effect of Ethanol on Cell Growth of Budding Yeast: Genes That Are Important for Cell Growth in the Presence of Ethanol

The budding yeast Saccharomyces cerevisiae has been used in the fermentation of various kinds of alcoholic beverages. But the effect of ethanol on the cell growth of this yeast is poorly understood. This study shows that the addition of ethanol causes a cell-cycle delay associated with a transient dispersion of F-actin cytoskeleton, resulting in an increase in cell size. We found that the tyrosine kinase Swe1, the negative regulator of Cdc28-Clb kinase, is related to the regulation of cell growth in the presence of ethanol. Indeed, the increase in cell size due to ethanol was partially abolished in the SWE1-deleted cells, and the amount of Swe1 protein increased transiently in the presence of ethanol. These results indicated that Swe1 is involved in cell size control in the presence of ethanol, and that a signal produced by ethanol causes a transient up-regulation of Swe1. Further we investigated comprehensively the ethanol-sensitive strains in the complete set of 4847 non-essential gene deletions and identified at least 256 genes that are important for cell growth in the presence of ethanol.

GSK‐3 kinase Mck1 and calcineurin coordinately mediate Hsl1 down‐regulation by Ca2+ in budding yeast

The Ca2+‐activated pathways of Saccharomyces cerevisiae induce a delay in the onset of mitosis through the activation of Swe1, a negative regulatory kinase that inhibits the Cdc28–Clb complex. Calcineurin and Mpk1 activate Swe1 at the transcriptional and post‐translational level, respectively, and both pathways are essential for the cell cycle delay. Our genetic screening identified the MCK1 gene, which encodes a glycogen synthetase kinase‐3 family protein kinase, as a component of the Ca2+ signaling pathway. Genetic analyses indicated that Mck1 functions downstream of the Mpk1 pathway and down‐regulates Hsl1, an inhibitory kinase of Swe1. In medium with a high concentration of Ca2+, Hsl1 was delocalized from the bud neck and destabilized in a manner dependent on both calcineurin and Mck1. Calcineurin was required for the dephosphorylation of autophosphorylated Hsl1. The E3 ubiquitin ligase complex SCFCdc4, but not the anaphase‐promoting complex (APC), was essential for Hsl1 destabilization. The Ca2+‐activated pathway may play a role in the rapid inactivation of Hsl1 at the cell cycle stage(s) when APC activity is low.

Identification of Saccharomyces cerevisiae Isoleucyl-tRNA Synthetase as a Target of the G1-specific Inhibitor Reveromycin A

To dissect the action mechanism of reveromycin A (RM-A), a G1-specific inhibitor, aSaccharomyces cerevisiae dominant mutant specifically resistant to RM-A, was isolated from a strain in which the genes implicated in nonspecific multidrug resistance had been deleted. The mutant gene (YRR2–1) responsible for the resistance was identified as an allele of the ILS1 gene encoding tRNAIle synthetase (IleRS). The activity of IleRS, but not several other aminoacyl-tRNA synthetases examined in wild type cell extract, was highly sensitive to RM-A (IC50 = 8 ng/ml). The IleRS activity of the YRR2–1 mutant was 4-fold more resistant to the inhibitor compared with that of wild type. The mutation IleRSN660D, near the KMSKS consensus sequence commonly found in the class I aminoacyl transferases, was found to be responsible for RM-A resistance. Moreover, overexpression of theILS1 gene from a high-copy plasmid conferred RM-A resistance. These results indicated that IleRS is a target of RM-Ain vivo. A defect of the GCN2 gene led to decreased RM-A resistance. IleRS inhibition by RM-A led to transcriptional activation of the ILS1 gene viathe Gcn2-Gcn4 general amino acid control pathway, and this autoregulation seemed to contribute to RM-A resistance.

Physiological Roles of Calcineurin in Saccharomyces cerevisiae with Special Emphasis on Its Roles in G2/M Cell-Cycle Regulation

Calcineurin, a highly conserved Ca2+/CaM-dependent protein phosphatase, plays key regulatory roles in diverse biological processes from yeast to humans. Genetic and molecular analyses of the yeast model system have proved successful in dissecting complex regulatory pathways mediated by calcineurin. Saccharomyces cerevisiae calcineurin is not essential for growth under laboratory conditions, but becomes essential for survival under certain stress conditions, and is required for stress-induced expression of the genes for ion transporters and cell-wall synthesis. Yeast calcineurin, in collaboration with a Mpk1 MAP kinase cascade, is also important in G2 cell-cycle regulation due to its action in a checkpoint-like mechanism. Genetic and molecular analysis of the Ca2+-dependent cell-cycle regulation has revealed an elaborate mechanism for the calcineurin-dependent regulation of the G2/M transition, in which calcineurin multilaterally activates Swe1, a negative regulator of the Cdc28/Clb complex, at the transcriptional, posttranslational, and degradation levels.

Fission yeast Mor2/Cps12, a protein similar to Drosophila Furry, is essential for cell morphogenesis and its mutation induces Wee1-dependent G2 delay

Fission yeast cells identify growing regions at the opposite ends of the cell, producing the rod‐like shape. The positioning of the growth zone(s) and the polarized growth require CLIP170‐like protein Tip1 and the Ndr kinase Orb6, respectively. Here, we show that the mor2/cps12 mutation disrupts the localization of F‐actin at the cell ends, producing spherical cells and concomitantly inducing a G2 delay at 36°C. Mor2 is important for the localization of F‐actin at the cell end(s) but not at the medial region, and is essential for the restriction of the growth zone(s) where Tip1 targets. Mor2 is homologous to the Drosophila Furry protein, which is required to maintain the integrity of cellular extensions, and is localized at both cell ends and the medial region of the cell in an actin‐dependent fashion. Cellular localization of Mor2 and Orb6 was interdependent. The tyrosine kinase Wee1 is necessary for the G2 delay and maintenance of viability of the mor2 mutant. These results indicate that Mor2 plays an essential role in cell morphogenesis in concert with Orb6, and the mutation activates the mechanism coordinating morphogenesis with cell cycle progression.

The involvement of the Saccharomyces cerevisiae multidrug resistance transporters Pdr5p and Snq2p in cation resistance.

The ATP‐binding cassette superfamily proteins Pdr5p and Snq2p of Saccharomyces cerevisiae are implicated in multidrug resistance. Here, we show that these transporters are also involved in cation resistance. Null mutants of PDR5 and SNQ2 genes exhibit increased sensitivity to NaCl, LiCl and MnCl2. The mutant cells grown in the presence of high concentrations of these metal salts contain higher levels of the metals than wild‐type cells. The expression of PDR5 and SNQ2 is induced by the metal salts. These results provide evidence that the yeast drug transporters contribute to cation resistance by regulating cellular cation homeostasis under ionic stress conditions.

Mutational analysis of the yeast multidrug resistance ABC transporter Pdr5p with altered drug specificity.

Multidrug resistance ABC transporter Pdr5p of Saccharomyces cerevisiae is particularly important due to its ability to export a wide range of unrelated substrates. To clarify its function, we generated Pdr5p mutants by random mutagenesis and screened for mutants with altered drug specificity in vivo by using 5 drug compounds. Nine point mutations that caused significant changes in drug specificity distributed throughout the length of Pdr5p, namely, in the extracellular, transmembrane or cytoplasmic regions of the transporter. We then investigated their effects upon drug resistance, using 36 chemically related or distinct substrates. From this study, overall geometry of the Pdr5p was suggested to contribute in acquiring the enormous range of drug specificity. Based on their ability to inhibit the growth of the mutant strains, the 36 tested drugs were classified into: drugs to which the mutants responded differently (Group 1), drugs to which all the mutants showed sensitivity (Group 2), and drugs to which all the mutants exhibited resistance (Group 3). The ability of the compounds to be partitioned to the plasma membrane seemed an important factor for recognition by Pdr5p.

Involvement of calcineurin-dependent degradation of Yap1p in Ca2+-induced G2 cell-cycle regulation in Saccharomyces cerevisiae

The Ca2+‐activated pathways in Saccharomyces cerevisiae induce a delay in the onset of mitosis through the activation of Swe1p, a negative regulatory kinase that inhibits the Cdc28p/Clb complex. We isolated the YAP1 gene as a multicopy suppressor of calcium sensitivity owing to the loss of ZDS1, a negative regulator of SWE1 and CLN2 gene expression. YAP1 deletion on a zds1Δ background exacerbated the Ca2+‐related phenotype. Yap1p was degraded in a calcineurin‐dependent manner when cells were exposed to calcium. In yap1Δ cells, the expression level of the RPN4 gene encoding a transcription factor for the subunits of the ubiquitin–proteasome system was diminished. The deletion of YAP1 gene or RPN4 gene led to the accumulation of Swe1p and Cln2p. Yap1p was a substrate of calcineurin in vivo and in vitro. The calcineurin‐mediated Yap1p degradation seems to be a long adaptive response that assures a G2 delay in response to a stress that causes the activation of the calcium signalling pathways.

Natural thioallyl compounds increase oxidative stress resistance and lifespan in Caenorhabditis elegans by modulating SKN-1/Nrf

Identification of biologically active natural compounds that promote health and longevity, and understanding how they act, will provide insights into aging and metabolism, and strategies for developing agents that prevent chronic disease. The garlic-derived thioallyl compounds S-allylcysteine (SAC) and S-allylmercaptocysteine (SAMC) have been shown to have multiple biological activities. Here we show that SAC and SAMC increase lifespan and stress resistance in Caenorhabditis elegans and reduce accumulation of reactive oxygen species (ROS). These compounds do not appear to activate DAF-16 (FOXO orthologue) or mimic dietary restriction (DR) effects, but selectively induce SKN-1 (Nrf1/2/3 orthologue) targets involved in oxidative stress defense. Interestingly, their treatments do not facilitate SKN-1 nuclear accumulation, but slightly increased intracellular SKN-1 levels. Our data also indicate that thioallyl structure and the number of sulfur atoms are important for SKN-1 target induction. Our results indicate that SAC and SAMC may serve as potential agents that slow aging.